The Arabidopsis male-sterile mutant, opr3, lacks the 12-oxophytodienoic acid reductase required for jasmonate synthesis

Abstract

Jasmonic acid (JA) and its precursor 12-oxophytodienoic acid (OPDA) act as plant growth regulators and mediate responses to environmental cues. To investigate the role of these oxylipins in anther and pollen development, we characterized a T-DNA-tagged, male-sterile mutant of Arabidopsis, opr3. The opr3 mutant plants are sterile but can be rendered fertile by exogenous JA but not by OPDA. Cloning of the mutant locus indicates that it encodes an isozyme of 12-oxophytodienoate reductase, designated OPR3. All of the defects in opr3 are alleviated by transformation of the mutant with an OPR3 cDNA. Our results indicate that JA and not OPDA is the signaling molecule that induces and coordinates the elongation of the anther filament, the opening of the stomium at anthesis, and the production of viable pollen. Just as importantly, our data demonstrate that OPR3 is the only isoform of OPR capable of reducing the correct stereoisomer of OPDA to produce JA required for male gametophyte development.

Figure 1

The biosynthesis of jasmonic acid from linolenic acid. The projections shown represent the absolute stereoconfiguration of the side chains.

Figure 2

Phenotypes of wild-type and opr3 mutant flowers. (A) Flowers at stage 12. (B) Flowers at anthesis. Pollination did not occur in opr3 plants unless buds had previously been treated with jasmonate (Bottom). (C) Germination of pollen harvested at anthesis.

Figure 3

Genome structure of the opr3 locus. The T-DNA (not to scale) with an additional 15 bp adjacent to the left border is inserted into a Tnat1 sequence (shaded), which is part of an intron in the OPR3 gene of ecotype WS. The four exons of OPR3 are shown, as are the BamHI sites used for plasmid recovery. The structure of the remainder of the T-DNA insert was not determined. The Col-O allele of OPR3 is shown for comparison.

Figure 4

Amino acid alignment of the A. thaliana OPR1 (GenBank accession number Y10617), OPR2 (U92460) and OPR3 (AF293653). Identical residues in at least two of three sequences are indicated in reverse print. The peroxisomal targeting signal in OPR3 is underlined.

Figure 5

Expression of OPR3 in anthers, flowers, and leaves of wild-type and opr3 plants. Gel-blot analysis was performed, using 10 μg of total RNA from anthers and 5 μg of poly(A)⁺ RNA from flowers and leaves, with an OPR3 cDNA as a probe. Ethidium bromide staining of the 18S rRNA band was used to confirm equal loading.